title: Target optimization for peptide nucleic acid (PNA)-mediated antisense inhibition of the CmeABC multidrug efflux pump in Campylobacter jejuni.
abstract: OBJECTIVE
  CmeABC is a resistance-nodulation-cell division (RND)-type multidrug efflux pump conferring resistance to clinically important antibiotics in Campylobacter. This study aimed to identify the optimal target sites for the inhibition of CmeABC with antisense peptide nucleic acid (PNA).
  
  
  METHODS
  Eighteen PNAs were designed to bind to the translational initiation regions of cmeABC, spanning the ribosome-binding site (RBS) and the start codon of the cmeABC genes. Campylobacter jejuni was treated with CmeABC-specific PNAs (CmeABC-PNAs) at various concentrations and subjected to western blotting to measure changes in the level of CmeABC expression. The MICs of ciprofloxacin and erythromycin were measured to evaluate the impact of CmeABC knockdown on antibiotic susceptibility.
  
  
  RESULTS
  While antisense PNA significantly affected CmeA and CmeB expression, interestingly, CmeC expression was not altered by any of the CmeC-PNAs used in this study. A CmeA-PNA targeting the RBS of cmeA and its upstream region reduced CmeA expression most efficiently, and CmeB expression was most significantly decreased by PNA binding to the RBS of cmeB and its downstream region. CmeA- and CmeB-PNAs increased the susceptibility of C. jejuni to ciprofloxacin and erythromycin in proportion to the inhibition levels observed in western blotting.
  
  
  CONCLUSIONS
  The cmeA gene is the best target to knockdown CmeABC with antisense PNA. The RBS is the major target for the PNA-mediated antisense inhibition of CmeABC. However, regions in its vicinity also significantly influence the effectiveness of the PNA-based knockdown of CmeABC.
authors.affiliation: School of Public Health, University of Alberta, Edmonton, Alberta, Canada.
authors.forename: Euna
authors.initials: E
authors.lastname: Oh
authors.forename: Qijing
authors.initials: Q
authors.lastname: Zhang
authors.forename: Byeonghwa
authors.initials: B
authors.lastname: Jeon
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D001483:Base Sequence; D016123:Campylobacter jejuni / Q000235:genetics* / Q000302:isolation & purification; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods / Q000639:trends*; D019450:Genes, MDR / Q000235:genetics*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D008969:Molecular Sequence Data; D020135:Peptide Nucleic Acids / Q000235:genetics*
chemical_list: D018528:ATP-Binding Cassette Transporters; D020135:Peptide Nucleic Acids
keywords: efflux pump inhibitors; fluoroquinolones; macrolides